Biodegradable photoluminescent polymers

ABSTRACT

Biodegradable photoluminescent polymer (BPLP) which comprises an oligomer synthesized from a multifunctional monomer, a diol, and an amino acid by reacting (i) a multifunctional monomer comprising citric acid or triethyl citrate with (ii) a diol to form a reaction product, and further reacting the reaction product with (iii) an amino acid, wherein the amino acid is linked as a side group to the oligomer backbone. The BPLP of the present invention poses tunable fluorescence emission characteristics and are cell-compatible and biodegradable. The BPLP can serve as both implant materials and bioimaging probes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/962,476, filed Aug. 8, 2013, now U.S. Pat. No. 9,145,467, which is acontinuation of U.S. patent application Ser. No. 13/000,327, filed Apr.5, 2011, now U.S. Pat. No. 8,530,611, which is a national stage entryapplication of International Application No. PCT/US2009/047845, filedJun. 18, 2009, which claims priority of U.S. Application Ser. No.61/074,503, filed Jun. 20, 2008.

FIELD OF INVENTION

The present invention relates in general to the field of biodegradablepolymers, and more particularly to the discovery and manufacture of anovel biodegradable photoluminescent polymer (BPLP) for biomedicalapplications.

BACKGROUND ART

Without limiting the scope of the invention, its background is describedin connection with applications and methods for manufacturingphotoluminescent polymers and the use of these polymers in thebiomedical fields.

US Patent Application No. 2002/0193522 (Yih-Min Sun, 2002) describes thesynthesis and luminescent characteristics of novel phosphorus containinglight-emitting polymers, especially one improving the luminescenceefficiency of the synthesis light-emitting polymers. According to themethod of the present invention, the electron-transporting chromophoresare introduced into an emission polymer to increase its electronaffinity. Further, several phosphorus-containing emission chromophoresare synthesized and incorporated with electron-transporting chromophoresfinally resulting in the novel phosphorus chromophores emitting bluelight as expected, improving thermal stability of resulting polymerssuch that the absorption peaks of these polymers are restricted to astable range.

WIPO Patent Application No: WO/2007/143209 (Fraser, et al. 2007)discloses luminescent diketonate polymers having fluorescent properties,phosphorescent properties, or both fluorescent and phosphorescentproperties.

U.S. Pat. No. 7,345,596 issued to Wallach and Lincoln, 2008 describessmart polymeric multilayer sensors in the form of beads suitable forsubmarine detection. The sensors have a change in detectable property,such as color, which occurs when said sensors are exposed to aparticular stimulus such as an object or event to be detected. Thechange in property is thus detectable by an external monitor.

US Patent Application No. 2002/0018843 A1 (Antwerp and Mastrotoaro,2002) discloses a method for determination of the concentration ofbiological levels of polyhydroxylated compounds, particularly glucose.The methods utilize an amplification system that is an analytetransducer immobilized in a polymeric matrix, where the system isimplantable and biocompatible. Upon interrogation by an optical system,the amplification system produces a signal capable of detection externalto the skin of the patient. Quantification of the analyte of interest isachieved by measurement of the emitted signal.

SUMMARY OF THE INVENTION

The present invention includes compositions and methods of making novelaliphatic biodegradable photoluminescent polymers (BPLPs) and theirassociated crosslinked variants (CBPLPs) for biomedical applications.The BPLPs are degradable oligomers synthesized from biocompatiblemultifunctional monomers including, e.g., citric acid, aliphatic diols,and various amino acids via a convenient and cost-effectivepolycondensation reaction. BPLPs can be crosslinked into elastomericcrosslinked polymers, CBPLPs. The present invention includes BPLP linkedto amino acids (e.g., (BPLP-cysteine (BPLP-Cys) and BPLP-serine(BPLP-Ser)), which offer advantages over traditional fluorescent organicdyes and quantum dots due to their preliminarily demonstratedcytocompatibility in vitro, minimal chronic inflammatory responses invivo, controlled degradability and high quantum yields, tunablefluorescence emission, and photostability.

One embodiment of the present invention describes an aliphaticbiodegradable photoluminescent polymer (BPLP) composition comprising adegradable oligomer, synthesized from a biocompatible multifunctionalmonomer, a diol; and an amino acid linked as a side chain to the BPLPbackbone. In one aspect the BPLP is optionally post-polymerized by acondensation reaction to form a crosslinked variant (CBPLP). Thebiocompatible monomer comprises citric acid, the diol comprises1,8-octanediol, and the amino acid comprises cysteine or serine. In oneaspect of the present invention the biocompatible monomer comprisescitric acid and the diols comprises saturated aliphatic diols, C3-C12diols, macrodiols, hydrophilic diols, hydrophobic diols or anycombinations thereof. In another aspect the diols are selected from agroup comprising of 1,8-octanediols, ethylene glycol, propylene glycol,macrodiols, poly(ethylene glycol), poly(propylene glycol)1,3-propanediol, ethanediol, and cis-1,2-cyclohexanediol. In yet anotheraspect the citric acid is at least partially replaced by unsaturatedmaleic acid or maleic anhydride, fumaric acid, fumaryl chloride,acroylchloride, itaconic acid, and allylmalonic acid monomers to yieldan injectable BPLP. In one aspect the BPLP is optionally crosslinked,wherein the crosslinking is achieved by radical polymerization initiatedby photoinitiators or redox initiators. The amino acids in the inventionare selected from alanine, arginine, asparagine, aspartic acid,cysteine, glycine, glutamine, glutamic acid, histidine, isoleucine,leucine, lysine, methionione, proline, phenylalanine, serine, threonine,tyrosine, tryptophan, valine or any combinations thereof.

In certain aspects the post-polymerization is a polycondensation or afree radical polymerization. The BPLP or the CBPLP of the presentinvention exhibits a fluorescence wherein the fluorescence emanates froma 6-membered aliphatic and biodegradable ring formed by a carboxylicacid, an alpha carbon, and an amino group of the amino acid. Thecarboxylic acid, the alpha carbon, and the amino groups bend backwardsto join the polymer backbone via an esterification reaction.

Another embodiment of the present invention describes a method of makingan aliphatic biodegradable photoluminescent polymer (BPLP) comprisingthe steps of: (i) mixing a biocompatible multifunctional monomer, adiol, and an amino acid to form a mixture, (ii) raising the temperatureof the mixture to melt the mixture, and (iii) lowering the temperatureof the mixture with stirring to form the aliphatic BPLP. The method ofthe present invention further comprises the step of purifying the BPLPby precipitating the BPLP in a solvent mixture or by dialysis. In oneaspect the biocompatible monomer used in the method described in thepresent invention comprises citric acid, the diol comprises1,8-octanediol, and the amino acid comprises cysteine or serine. Thesolvent mixture used in the method of the present invention is selectedfrom a group comprising of 1,4-dioxane/water, ethanol/water,acetone/water, and tetrahydrofuran/water.

In the method of the present invention the biocompatible multifunctionalmonomer and the diol are provided in equal amounts and the biocompatiblemonomer comprises maleic acid, unsaturated monomers, or citric acid atleast partially replaced with unsaturated maleic acid, maleic anhydride,fumaric acid, fumaryl chloride, acroylchloride, itaconic acid, andallylmalonic acid. In yet another aspect the BPLP is injectable.

In yet another embodiment the present invention describes a method ofmaking a cross-linked biodegradable photoluminescent polymer (CBPLP)comprising the steps of: dissolving a biodegradable photoluminescentpolymer in an organic solvent to form a solution, casting the solutionin a mold, evaporating the solvent, and post-polymerizing the BPLP toforms the CBPLP. The organic solvent used in the manufacture of theCBPLP is selected from a group comprising of 1,4-dioxane, ethanol,acetone, and tetrahydrofuran.

The present invention also discloses a method of making a water solublealiphatic biodegradable photoluminescent polymer (BPLP) comprising thesteps of: (i) mixing equal amounts of citric acid and polyethyleneglycol to form a mixture, (ii) adding serine or cysteine to the mixture,(iii) raising the temperature of the mixture to melt the mixture, and(iv) lowering the temperature of the mixture while stirring to form thewater soluble BPLP.

In one embodiment the present invention further describes a method ofmaking one or more aliphatic biodegradable photoluminescent polymer(BPLP) nanoparticles comprising the steps of: dissolving a BPLP in anorganic solvent to form a solution, adding the solution dropwise todeionized water with stirring, and evaporating the organic solvent toform the one or more aliphatic (BPLP) nanoparticles. In one aspect theBPLP comprises a degradable oligomer synthesized from a biocompatiblemultifunctional monomer, a diol, and an amino acid, wherein thebiocompatible monomer comprises citric acid the diols comprisessaturated aliphatic diols, C3-C12 diols, macrodiols, hydrophilic diols,hydrophobic diols or any combinations thereof. In another aspect theamino acids are selected from alanine, arginine, asparagine, asparticacid, cysteine, glycine, glutamine, glutamic acid, histidine,isoleucine, leucine, lysine, methionione, proline, phenylalanine,serine, threonine, tyrosine, tryptophan, and valine or any combinationsthereof. In yet another aspect the one or more aliphatic (BPLP)nanoparticles are fluorescent, wherein the fluorescence emanates from a6-membered aliphatic and biodegradable ring formed by a carboxylic acid,an alpha carbon, and an amino group of the amino acid. The carboxylicacid, the alpha carbon, and the amino groups bend backwards to join thepolymer backbone via an esterification reaction. In a certain aspect the6-membered ring is a ester-amide ring

One embodiment of the present invention is directed towards anurethane-doped biodegradable photoluminescent polyester (UBPLP)composition comprising: a degradable oligomer, wherein the oligomer issynthesized from a biocompatible multifunctional monomer, a diol, anamino acid, and a di-isocyanate wherein the amino acid is linked as aside chain to the BPLP backbone. The biocompatible monomer in the CUBPLPof the present invention further comprises citric acid, the diolcomprises 1,8-octanediol, and the amino acid comprises cysteine orserine and the di-isocyanate comprises Hexamethylene-1,6-di-isocyanate(HDI), or 1,4-Butane di-isocyanate (BDI). In one aspect of the presentinvention the biocompatible monomer comprises citric acid. In anotheraspect the diols comprises saturated aliphatic diols, C3-C12 diols,macrodiols, hydrophilic diols, hydrophobic diols or any combinationsthereof. In yet another aspect the diols are selected from a groupcomprising of 1,8-octanediols, ethylene glycol, propylene glycol,macrodiols, poly(ethylene glycol), poly(propylene glycol)1,3-propanediol, ethanediol, and cis-1,2-cyclohexanediol. The aminoacids in the CUBPLP are selected from alanine, arginine, asparagine,aspartic acid, cysteine, glycine, glutamine, glutamic acid, histidine,isoleucine, leucine, lysine, methionione, proline, phenylalanine,serine, threonine, tyrosine, tryptophan, valine or any combinationsthereof. The di-isocyanates are selected from a group comprisingHexamethylene-1,6-di-isocyanate (HDI), 1,4-Butane di-isocyanate (BDI),lysine di-isocyanate (LDI), or any other di-isocyanates. In a specificaspect the UBPLP exhibits a fluorescence emanating from a 6-memberedaliphatic and biodegradable ring formed by a carboxylic acid, an alphacarbon, and an amino group of the amino acid. The carboxylic acid, thealpha carbon, and the amino groups bend backwards to join the polymerbackbone via an esterification reaction.

In another embodiment the present invention describes a method of makingan a crosslinked urethane-doped biodegradable photoluminescent polyester(CUBPLP) comprising the steps of: (i) mixing a biocompatible monomer anda diol to form a mixture, (ii) raising the temperature of the mixture tomelt the mixture, (iii) lowering the temperature of the mixture withstirring to form an oligomer, (iv) adding an amino acid to the oligomerwith stirring to form a pre-BPLP-amino acid, (iv) purifying thepre-BPLP-amino acid by dropwise addition to deionized water, (v)collecting an undissolved pre-BPLP-amino acid portion from the deionizedwater, (vi) lyophilizing the collected pre-BPLP-amino acid to obtainpurified pre-BPLP, (vii) dissolving the purified pre-BPLP-amino acid in1,4-dioxane to form a solution, (viii) adding 1,6-hexamethyldiisocyanate (HDI) to the pre-BPLP-amino acid solution which mayoptionally contain one or more catalysts to form a pre-CUBPLP (UBPLP),(ix) casting a film of the pre-CUBPLP (UBPLP) in a laminar airflow, and(x) placing the pre-CUBPLP (UBPLP) in an oven to obtain the CUBPLP. Thebiocompatible monomer used in the manufacture of the CUBPLP comprisescitric acid, the diol comprises 1,8-octanediol, and the amino acidcomprises cysteine or serine. In one aspect the one or more catalystsare selected from a group comprising tin-based catalysts such asorganotin catalysts, tin octanoate (stannous octanoate), dibutyl tindilaurate and amine catalysts such DABCO(1,4-diazabicyclo[2.2.2]octane).

In yet another embodiment the present invention describes a method forfabricating crosslinked aliphatic biodegradable photoluminescent polymer(CBPLP) scaffolds comprising the steps of: (i) freeze-drying a BPLPsolution in a mold and (ii) post polymerizing the freeze-dried solutionin an oven to form a CBPLP scaffold.

Yet another embodiment of the present invention is directed towards amethod of fabricating a small diameter blood vessel (SDBV) graft,wherein the SDBV graft comprises multiple crosslinked aliphaticbiodegradable photoluminescent polymer (CBPLP) scaffolds; comprising thesteps of: (i) transferring first cells on a first CBPLP scaffold, (ii)transferring second cells on a second CBPLP scaffold, (iii) culturingthe first and second CBPLP scaffolds for at least two days, (iv)providing a CBPLP-ser tube, (v) constructing the SDBV graft by rollingthe first CBPLP seeded with the first cell and the second CBPLP scaffoldseeded with the second cell with the CBPLP-ser tube sequentially on arod, (vi) removing the rod to form a tubular graft, (vii) seeding thirdcells onto the lumen of the tubular graft, (viii) culturing the graftfor at least 3 days in a coculture medium, and (ix) assembling the graftin a perfusion bioreactor to form the CBPLP-SDBV graft. The method offabricating a SDBV as described in the present invention furthercomprises the steps of: mounting the grafts on one or more hollow postsin the perfusion chamber, pumping a cell-culture medium with pulsinginto the chamber; wherein the cell-culture medium is in communicationwith the grafts, and culturing the grafts for a specified period of timein the cell-culture medium. In one aspect of the method the first cellsare human aortic fibroblast (HAFB) cells. In another aspect the secondcells are human aortic smooth muscle cells (HASMC) cells. In yet anotheraspect the third cells are human aortic endothelial cells (HAEC) cells.

In a specific embodiment the present invention details a fluorometricmethod for the detection of cationic polymers in a sample, wherein thecationic polymer does not have a chromophore or lacks a chromophoreabsorbing above 200 nm comprising the steps of: (i) mixing the samplecomprising the cationic polymer with an aqueous solution of awater-soluble biodegradable photoluminescent polymer (BPLP) or anorganic solvent of a water-insoluble BPLP, (ii) forming a complexbetween the cationic polymer in the sample and the water-soluble orwater-insoluble BPLP, (iii) measuring a fluorescence signal emanatingfrom the complex of the cationic polymer with the water-soluble orwater-insoluble BPLP, (iv) mixing standard solutions comprisingdifferent concentrations of the cationic polymer with the with thewater-soluble or water-insoluble BPLP solution, (v) forming complexesbetween the different concentrations of the cationic polymer and thewater-soluble or water-insoluble BPLP, (vi) measuring a fluorescencesignal emanating from the complex of the different concentrationscationic polymer with the water-soluble or water-insoluble BPLP, (vii)creating a calibration curve by plotting the fluorescence signal valuesof the complexes versus the different concentrations of the cationicpolymer, and (viii) calculating an unknown concentration of the cationicpolymer in the sample based on the calibration curve. In one aspect thecationic polymer isPolyquaternium-1,ω-{4-[tris(2-hydroxyethyl)ammonio]-but-2-enylpoly(dimethylammoniobut-2enyl)}tris(2-hydroxyethyl)ammoniumpolychloride. In another aspect the fluorescence signal is proportionalto the concentration of the cationic polymer in the solution.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures and in which:

FIG. 1 is a schematic which shows the steps involved in the synthesis ofBPLP-Cys;

FIG. 2A shows the FTIR spectra of BPLP-Cys (POC-Cys) as a representativeBPLP;

FIG. 2B shows the ¹H-NMR spectra of BPLP-Cys as a representative BPLP;

FIG. 2C shows the ¹³C-NMR spectra of BPLP-Cys as a representative BPLP;

FIG. 3A shows the MALDI-MS spectra used to determine the molecularweight of BPLP-Cys 0.2 (the number average molecular weight was 1334Da);

FIG. 3B shows the MALDI-MS spectra used to determine the molecularweight of BPLP-Ser 0.2 (the number average molecular weight was 1459Da);

FIG. 4A shows the photoluminescence (PL) emission spectra of BPLP-Cyssolution in 1,4-dioxane with various molar ratios of L-cysteine excitedat 350 nm;

FIG. 4B shows the PL emission spectra of CBPLP-Cys film with variousmolar ratios of L-cysteine excited at 350 nm;

FIG. 4C shows the PL excitation and emission spectra of BPLP-Cys 0.2porous scaffold);

FIG. 4D shows the PL excitation and emission spectra of BPLP-Cys-0.2nanoparticles (average diameter is 80 nm) (various forms of BPLP-Cys allemit strong fluorescence);

FIG. 4E shows the PL emission spectra of BPLP-serine-0.2 (BPL-Ser-0.2);

FIG. 4F shows the photostability evaluation of BPLP-Cys0.2 solution andfilm, BPLP-Ser0.2 solution and control organic dye Rhodamine B;

FIG. 4G shows the intensity-absorbance curve of BPLP-Cys for quantumyield measurements;

FIG. 4H shows the PL emission spectra of BPLP-Cys, POC and all themonomers used for BPLP-Cys synthesis;

FIG. 5A shows the fluorescence intensity changes over mass remaining forBPLP-Cys0.2 exposed to 0.05M NaOH;

FIG. 5B shows the molecular weight changes over mass remaining forBPLP-Cys0.2 exposed to 0.05M NaOH;

FIG. 6A shows the in vitro degradation of BPLP-Cys in PBS (pH=7.4) at37° C. (n=5);

FIG. 6B shows the in vitro degradation of CBPLP-Cys in PBS (pH=7.4) at37° C. (n=5);

FIG. 6C shows the tensile strength and initial Young's modulus ofCBPLP-Cys synthesized with various molar concentration of L-cysteine(n=5);

FIG. 6D shows the elongation of CBPLP-Cys synthesized with various molarconcentration of L-cysteine (n=5);

FIG. 7A shows the HPLC-ESI-MS analysis of soluble in vitro degradationproducts for BPLP-Cys degraded in 0.05 M NaOH for 24 hours;

FIG. 7B shows the HPLC-ESI-MS analysis of soluble in vitro degradationproducts for BPLP-Ser degraded in 0.05 M NaOH for 24 hours;

FIG. 8A shows the cell viability and proliferation assay (MTT assay) for3T3 fibroblasts cultured on BPLP-Cys film with POC and PLGA used ascontrols;

FIG. 8B shows the cytotoxicity evaluation of degradation products ofBPLPs (-Cys and -Ser) and CBPLPs (-Cys and -Ser) at 2×, 10×, 50× and100× dilutions with POC and PLGA75/25 used as controls (all data werenormalized to the mean absorbance of PLGA (100× dilution));

FIG. 8C shows BPLP-Ser nanoparticle-uptaken 3T3 fibroblasts observedunder the light microscope (20×), scale bar is 100 μm, with an inset TEMpicture of BPLP-Ser nanoparticles (80 nm);

FIG. 8D shows BPLP-Ser nanoparticle-uptaken 3T3 fibroblasts observedunder fluorescent microscope with FITC filter (20×), scale bar is 100μm;

FIG. 8E shows BPLP-Ser nanoparticle-uptaken 3T3 fibroblasts observedunder fluorescent microscope with Texas Red filter (20×), scale bar is100 μm;

FIG. 8F shows the fluorescence image of BPLP-Ser nanoparticles injectedsubcutaneously in a nude mouse;

FIG. 8G shows the SEM picture of the cross section of a porous BPLP-Serscaffold, scale bar is 200 μm;

FIG. 8H shows the fluorescence image of BPLP-Ser porous scaffoldimplanted subcutaneously in a nude mouse;

FIG. 9 shows the H&E staining image (100×) for BPLP-Ser0.2 scaffoldsimplanted into nude mice for 5 months;

FIG. 10A shows photoluminescent properties of BPLP-Thr0.2 stored inamber glass bottles at −20° C. for over a year;

FIG. 10B shows photoluminescent properties of BPLP-Leu0.2 stored inamber glass bottles at −20° C. for over a year;

FIG. 11 shows the excitation and emission spectra of UBPLP-Cys;

FIG. 12 shows the tensile strength and initial Young's modulus ofCUBPLP-Cys0.2 with different thermo-crosslinking condition;

FIG. 13 shows the elongation of CUBPLP-Cys0.2 with differentthermo-crosslinking condition;

FIG. 14 shows the excitation and emission spectra ofWBPLP-PEG200-Cys0.2;

FIG. 15 shows the excitation and emission spectra ofWBPLP-PEG200-Ser0.2;

FIG. 16 shows the excitation and emission spectra ofPCBPLP-MA0.8-Cys0.2;

FIG. 17A shows the tubes fabricated from the BPLP of the presentinvention under natural light;

FIG. 17B shows the tubes fabricated from the BPLP of the presentinvention under UV light;

FIG. 18 is a schematic representation of BPLP scaffold-sheet strategyfor blood vessel tissue engineering; and

FIG. 19 shows a chemical structure of Polyquaternium-1.

DESCRIPTION OF THE INVENTION

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts thatcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention.

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

The present invention describes the development of novel aliphaticbiodegradable photoluminescent polymers (BPLPs) and their associatedcrosslinked variants (CBPLPs) for biomedical applications. The BPLPs ofthe present invention are degradable oligomers synthesized frombiocompatible multifunctional monomers including citric acid, aliphaticdiols, and various amino acids via a convenient and cost-effectivepolycondensation reaction. The term “multifunctional monomers” as usedherein describe monomers including citric acid and its derivatives suchas triethyl citrate. The multifunctional monomer such as citric acid canbe partially replaced by unsaturated dicarboxyl monomers to makeunsaturated BPLPs. The unsaturated carboxylic monomers include but notlimited to vinyl-containing maleic acid, maleic anhydride, fumaric acid,fumaryl chloride, and acroylchloride which have at least onecarbon-carbon double bonds. The unsaturated BPLPs can be polymerized orcrosslinked via radical polymerizations initiated by photoinitiatorsand/or redox initiators. The unsaturated BPLPs can be furthercopolymerized or crosslinked with other vinyl-containing monomers suchas acrylic monomers. Non-limiting examples of vinyl-containing monomersinclude acrylates and methacrylates, i.e., diacrylates, triacrylates,dimethacrylates, and trimethacrylates, multifunctional allyliccompounds, such as diallyl maleate and allyl methacrylate,multifunctional monomers having a vinyl functionality are also included,allyl methacrylate (AMA), diallyl maleate (DAM), divinyl benzene (DVB),ethylene glycol dimethacrylate (EGDMA), N,N′-methylene-bis-acrylamide(NNMBA), tripropylene glycol diacrylate (TPGDA), triallyl cyanurate(TAC), triethylene glycol dimethacrylate (TEDMA, TEGMA),trimethylolpropane triacrylate (TMPTA), trimethylolpropanetrimethacrylate (TMPTMA, TRIM), and trimethylolpropane diallyl ether(TMPDAE). The diols comprises saturated aliphatic diols includingsaturated diols such as C3-C12 diols, macrodiols, hydrophilic diols,hydrophobic diols or any combinations thereof, or unsaturated diols suchas cis-2-butene-1,4-diol. The amino acids can be L-amino acids, D-aminoacids, D,L-amino acids and their derivatives and combinations. BPLPs canbe further crosslinked into elastomeric crosslinked polymers, CBPLPs.The inventors in the present disclosure show representatively thatBPLP-cysteine (BPLP-Cys) and BPLP-serine (BPLP-Ser) offer advantagesover the traditional fluorescent organic dyes and quantum dots due totheir preliminarily demonstrated cytocompatibility in vitro, minimalchronic inflammatory responses in vivo, controlled degradability andhigh quantum yields (up to 62.33%), tunable fluorescence emission (up to725 nm), and photostability.

The tensile strength of CBPLP-Cys film of the present invention rangedfrom 3.25±0.13 MPa to 6.5±0.8 MPa and the initial modulus was in therange of 3.34±0.15 MPa to 7.02±1.40 MPa. Elastic CBPLP-Cys could beelongated up to 240±36%. The compressive modulus of BPLP-Cys (0.6)(1:1:0.6 OD:CA:Cys) porous scaffold was 39.60±5.90 KPa confirming thesoft nature of the scaffolds. In addition the BPLPs described alsopossessed great processability for micro/nano-fabrication. The inventorshave further shown that the BPLP-Ser nanoparticles (“biodegradablequantum dots”) can be used for in vitro cellular labeling andnon-invasive in vivo imaging of tissue engineering scaffolds.

None of the current biodegradable polymers can function as both implantmaterials and fluorescent imaging probes. The development of BPLPs andCBPLPs as described in the present invention represents a new directionin developing fluorescent biomaterials and could impact tissueengineering, drug delivery, bioimaging.

A novel biomaterial may create new fields of study and opportunities totackle unmet scientific problems. The discovery of fluorescent quantumdots is a good example.¹⁻⁴ The unique photoluminescent properties offluorescent quantum dots brings tremendous opportunities for cancertherapy and diagnosis through biological labeling and imaging.Similarly, fluorescent protein has become one of the most importanttools in bioscience, since it can reveal processes previously invisible.Fluorescent biomaterials have been an intense research focus inbiomedical and biological fields with wide applications in cellularimaging, biosensing, immunology, drug delivery and tissueengineering.⁵⁻¹⁰ Current fluorescent biomaterials include fluorescentorganic dyes, fluorescent proteins, lanthanide chelates, and quantumdots. Most of the organic dyes such as fluoresceins, rhodamines, andcyanine dyes are not used in vivo because they exhibit poorphotostability and substantial cytotoxicity.^(11, 12) Fluorescentproteins often suffer from photobleaching^(13, 14) and low quantumyield.¹⁵ Furthermore, the aggregation of fluorescent proteins insidecells may cause cellular toxicity.¹⁶ Although various surfacemodifications have been attempted to reduce theirtoxicity,^(9, 12, 17, 18) the accumulation of toxic ions released fromquantum dots remains a significant concern, especially for long-term usein vivo.

Synthetic fluorescent polymers have been developed for variousnon-biological applications, such as light emitting diodes.¹⁹ Thesepolymers are not degradable and usually contain conjugated phenyl unitsraising concerns of potential carcinogenesis or toxicity when used forin vivo biomedical applications. Hitherto, biodegradable fluorescentpolymers have required conjugation or encapsulation of the organic dyesor quantum dots on or in the degradable polymers in order to bevisualized.^(11, 20-23) However, these approaches do not address thepreviously mentioned drawbacks of the organic dyes and quantum dots.Thus, there is an urgent need for the development of biodegradable andbiocompatible photoluminescent materials.

The present invention reports the development of aliphatic biodegradablesynthetic polymers, which show intriguing photoluminescence phenomena. Aseries of novel biodegradable photoluminescent polymers, referred to asBPLPs are described in the present disclosure. BPLPs arelow-molecular-weight aliphatic oligomers that include both water-solubleand water-insoluble oligomers. They can be further processed to formelastomeric crosslinked BPLPs (CBPLPs), which not only possess desirablemechanical properties, but also retain strong, tunable fluorescenceemission ranging from blue to red. Tunability is afforded by theincorporation of different amino acid residues during polymer synthesis.CBPLPs can be used as implant or device materials and, in addition, asin vivo bioimaging probes. The present invention studies the in vitrocellular uptake of fluorescent BPLP nanoparticles and presents theresults of in vivo fluorescence bioimaging of CBPLP scaffolds todemonstrate their potential use in cellular fluorescence labeling, drugdelivery and tissue engineering. The present disclosure provides furtherevidence related to the in vitro degradation and proffer a mechanismthrough which the photoluminescence of these promising materials areachieved.

Synthesis and Characterization of the BPLP Families:

The synthesis of BPLPs and CBPLPs are straightforward and similar tothat for the previously developed biodegradable elastomers,poly(octamethylene citrates) (POC).^(24, 25) POC is elastomericmaterials. POC porous scaffold is soft (compressive Young's modulus:0.482 MPa similar to that of many soft tissues in the body⁴⁰ and fullyelastic (100% recovery after 500 times of cyclic compression). For thesynthesis of POC, citric acid (CA) was reacted with 1,8-octanediol (OD)via a condensation reaction to form an oligomer referred to as pre-POC.The pre-POC was then post-polymerized through further condensation toform an elastomeric crosslinked polymer network. Similarly, any of thetwenty (enantiomerically-pure (L-)) amino acids were added into thereaction of citric acid and 1,8-octanediol to prepare a family ofoligomeric BPLPs such as BPLP-cysteine (BPLP-Cys or POC-Cys) andBPLP-serine (BPLP-Ser or POC-Ser). BPLPs can be optionally furtherpost-polymerized to form CBPLPs. BPLPs are soluble in organic solventssuch as 1,4-dioxane, ethanol, acetone, and tetrahydrofuran whenhydrophobic diols such as 1,8-octanediol were used. Water soluble BPLPscould be synthesized using hydrophilic diols such as poly(ethyleneglycol) (e.g. PEG 200 and PEG 400).

The proposed polymer structures are shown in FIG. 1A. The synthesis ofBPLPs is relatively simple. The typical synthesis is described asfollowing: Equimolar amount of citric acid reacted with 1,8-octanediolat 140° C. for 15 min after they were melted at 160° C. in around-bottom flask then L-cysteine (0.2, 0.4, 0.6 and 0.8 molar rationof cystein/citric acid) was added to the flask to stir for additional 60min at 140° C. to form a BPLP-cys (or POC-cys) as shown in FIG. 1A.BPLP-cys could be crosslinked via post-polymerization in an oven at 80°C. for 4 days to form crosslinked BPLP-cys (CBPLP-cys or CPOCcys) viathe still reactable side and end —COOH and —OH on BPLPcys.

BPLP-cys can be dissolved in multiple organic solvents such as1,4-dioxane, ethanol, acetone, tetrahydrofuran (THF) etc. Thedegradation studies of BPLP-cys and CBPLP-cys confirmed that bothpolymers are degradable polymers. Modulating the ratio ofLcysteine/citric acid and post-polymerization conditions can adjustdegradation of BPLPs and CBPLPs. When poly(ethylene glycol) (PEG, Mw 200or 400) was added to react with citric acid, water-soluble BPLP-PEG-cysis synthesized. The introduction of PEG was expected to speed updegradation. Thus, the inventors were able to synthesize bothwater-soluble and organic solvent soluble BPLPs, maximizing thepotential of using BPLPs for biological applications. For example,water-soluble BPLPs can be used as fluorescence dye like greenfluorescent protein (GFP) or any other water soluble dyes for biologicallabeling. Water-insoluble BPLPs could be used to fabricatenanoparticles, cell scaffolds for drug delivery and tissue engineeringapplications. The quantum yields of the BPLP-Cys (62.3%) and BPLP-Ser(26.0%) are much higher than those reported for fluorescent proteinssuch as green fluorescent protein (GFP) (7.3%) and its blue variants(7.9%) [26,27]. The decay lifetime of BPLP-cys is about 10 ns longerthan the common GFP (1.3-3.7 ns)[114, 115].

Polymer characterizations were conducted for BPLP-Cys as arepresentative BPLP, except where otherwise specified (FIGS. 2A to 2C).For Fourier Transform Infrared (FT-IR) analysis, purified BPLP wasdissolved in 1,4-dioxane to make a 5 wt % solution. The BPLP solutionwas cast onto KBr pellets and the solvent was evaporated overnight.FT-IR spectra were collected at room temperature using a Nicolet 6700FTIR spectrometer (Thermo Fisher Scientific). For nuclear magneticresonance (NMR) analysis, 5 mg of polymer was dissolved in 1 ml ofdeuterated dimethyl sulfoxide (DMSO-d₆). The ¹H-NMR was recorded at roomtemperature on a JEOL 300 MHz spectrometer, whereas the ¹³C-NMR wasrecorded on a JEOL 500 MHz spectrometer. Tetramethylsilane was used asinternal reference in both cases. For matrix-assisted laserdesorption/ionization mass spectroscopy (MALDI-MS) analysis, the polymerwas ionized with the use of a α-cyano cinnimateN,N-diisopropylethylammonium ionic liquid polymer matrix.

The FTIR spectra (FIG. 2A) confirmed the presence of —SH at 2575 cm⁻¹,—C(═O)NH— at 1527 cm⁻¹, —C═O at 1731 cm⁻¹, —CH₂— at 2931 cm⁻¹, and —OHat 3467 cm⁻¹. In the ¹H-NMR spectra of BPLP-Cys (FIG. 2B), the presenceof the peaks at 1.02 ppm (—CH₂SH from L-cysteine), 1.23 ppm and 1.50 ppm(—CH ₂— from 1,8-octanediol), and the multiple peaks at 2.75 ppm (—CH ₂—from citric acid) confirmed the incorporation of L-cysteine intopre-POC. In the ¹³C-NMR spectra of BPLP-Cys (FIG. 2C), the peaks around170 ppm were assigned to carbonyl (C═O) groups from citric acid andL-cysteine. The peaks around 63.8 ppm and 28.5 ppm were assignedrespectively to —O—CH₂CH₂— and —O—CH₂ CH₂— from 1,8-octanediol. The—C(═O)—CH₂— carbon from citric acid was assigned to the peak at 61.2ppm. The —HN—CH— carbon from L-cysteine was assigned to the peak at 54.5ppm. There were four peaks assigned to the central carbon atoms ofcitrate units in various chemical environments. Peaks at 72.9 and 73.4were assigned to C1 when R¹ is —(CH₂)₈—OH and —H respectively. Peaks at72.1 and 72.4 ppm were assigned to C2 and C3 respectively. However, the¹³C-NMR of pre-POC only showed two peaks of central C of citrate unitsat 72.9 and 73.4 ppm. The ¹³C-NMR results suggest the presence of a6-membered ring with conjugated character formed on BPLP-Cys as depictedin FIG. 1A. A 6-membered ring formed between L-cysteine and hydroxylgroups on the central C of the citrate unit is proposed to beresponsible for the fluorescence as discussed below. The averagemolecular weight of BPLP-Cys0.2 (formed by reaction of 1:1:0.2OD:CA:Cys) measured by MALDI-MS was 1334 Dalton (FIG. 3). The abovepolymer characterization confirmed that L-cysteine was incorporated intothe BPLP-Cys. The overall BPLP synthesis is believed to have resulted ina blend of oligomers of POC (pre-POC) and BPLP-Cys as shown in FIG. 1Adue to the low percentage of L-cysteine in the polymers.

Photoluminescence Properties of BPLPs and CBPLPs:

The various forms of BPLPs, including BPLP solution (FIG. 4A), CBPLPfilms (FIG. 4B), CBPLP scaffolds (FIG. 4C), and BPLP nanoparticles (FIG.4D), all emit strong fluorescence. The fluorescence intensity ofBPLP-Cys can be tuned by varying the molar concentration of L-cysteinein the polymers (FIG. 4A). FIG. 4E shows that BPLP-serine (BPLP-Ser)emits different fluorescent colors from blue to red depending on theexcitation wavelength. To further explore this class of material, theinventors synthesized a family of BPLPs using each of the 20 naturalamino acids. The BPLPs were found to exhibit fluorescence colors rangingfrom blue to red (up to 725 nm) (Table 1) depending on the choice ofamino acid.

All photoluminescence spectra were acquired on a Shimadzu RF-5301 PCfluorospectrophotometer. Both the excitation and the emission slitwidths were set at 1.5 nm for all samples unless otherwise stated. BPLPsolutions (5% w/w) in 1,4-dioxane were loaded in a quartz cuvette with apathlength of 10 mm. To collect the excitation and emission spectra forscaffolds and films (size of about 12 mm×40 mm), samples were helddiagonally in a quartz cuvette with a pathlength of 10 mm. The quantumyields of the BPLP polymers were measured by the Williams' method.Briefly, 5% BPLP-Cys 0.2 solution was prepared. The solution was scannedat various excitation wavelengths. Optimal excitation wavelength wasdetermined as the one which generated the highest emission intensity.Then, UV-vis absorbance spectrum was collected with the same solutionand the absorbance at the optimal excitation wavelength was noted. Then,a series of solution was prepared with gradient concentration, so thatthe absorbance of the each solution was within the range of 0.01-0.1 Absunits. The fluorescence spectrum was also collected for the samesolution in the 10 mm fluorescence cuvette. The fluorescence intensity,which is the area of the fluorescence spectrum, was calculated andnoted. Five solutions with different concentrations were tested and thegraphs of integrated fluorescence intensity vs. absorbance were plotted.The quantum yields of the BPLP polymers were calculated according toequation (1) where, Φ=quantum yield; Slope=gradient of the curveobtained from the plot of intensity versus absorbance; η=Refractiveindex of the solvent; x=subscript to denote the sample, and ST=subscriptto denote the standard.

$\begin{matrix}{\Phi_{X} = {{\Phi_{ST}\left( \frac{{Slope}_{X}}{{Slope}_{ST}} \right)}\left( \frac{\eta_{X}}{\eta_{ST}} \right)^{2}}} & (1)\end{matrix}$

Anthracene (quantum yield=0.27 in ethanol) was used as a standard. TheBPLP polymers were dissolved in 1,4-dioxane and anthracene was dissolvedin ethanol. The slit width was kept same for both standard and samples.Absorbance was measured using a SHIMADZU UV-2450 spectrophotometer.

The fluorescence intensity of BPLP-Cys decreased only slightly (<2%)after continuous UV excitation for 3 hrs indicating excellentphotostability as compared to the organic fluorescent dye rhodamine-B(FIG. 4F). The quantum yields of the BPLP-Cys (62.3%) and BPLP-Ser(26.0%) (FIG. 4G and Table 1) were much higher than those reported forfluorescent proteins such as green fluorescent protein (GFP) (7.3%) andits blue variants (7.9%).¹⁵ The emission range and quantum yields of allBPLPs are listed in Table 1.

TABLE 1 Range of excitation and emission wavelengths and quantum yieldsfor BPLPs with twenty different L-amino acids. BPLP-amino acid solutions(1% w/w in 1,4-dioxane) were used for photoluminescencecharacterization. Quantum BPLP- Exc (nm) Emi (nm) Yield (%) Ala 250-413295-524 5.3 Arg 250-503 297-594 0.9 Asn 280-490 299-623 11.0 Asp 275-415301-493 11.4 Cys 240-420 312-561 62.3 Glu 255-415 296-647 0.3 Gln280-500 296-647 13.9 Gly 265-510 295-678 10.9 His 310-540 330-650 1.9Ile 250-403 291-499 1.2 Leu 275-415 311-525 1.0 Lys 265-535 291-646 9.4Met 250-396 286-491 0.5 Phe 270-420 294-498 0.8 Pro 255-450 294-533 0.4Ser 290-660 303-725 26.0 Thr 250-470 313-580 34.2 Trp 300-490 340-58812.1 Tyr 240-440 311-561 3.1 Val 240-391 279-495 1.0

BPLP-Cys was degraded in 0.05 M NaOH solution. Fluorescence intensitywas based on the same concentration of BPLP-Cys in 1,4-dioxane atvarious degradation degrees. Molecular weight was determined byMALDI-MS. The fluorescence intensity of BPLP-Cys0.2 increased withincreasing degradation in NaOH solution (FIG. 5A). It should be notedthat the fluorescence measurements for polymers under degradation werebased on the same concentration of BPLP-Cys in 1,4-dioxane at variousdegrees of degradation. MALDI-MS analysis indicated that the molecularweight of the insoluble polymer did not significantly change duringdegradation in NaOH solution (FIG. 5B). This is due to the fact that thepolymers containing fluorescent ring-structures may degrade more slowlythan the polymers without the ring-structures (pre-POC) due to therelatively higher stability of the amide bonds in the ring-structures.Considering that the molecular weight of pre-POC (Mn=1088 Da)²⁵ is closeto that of Mw of the resulting BPLP-Cys which may contain pre-POC, thedegradation may result in an erosion on the pre-POC first, leavingbehind the low percentage of BPLP-Cys without significant molecularweight changes. Therefore, the polymer degradation is proposed to haveresulted in an increasing concentration of the polymer chains with thefluorescent ring-structures.

Exploration of the Fluorescence Mechanism:

The potential mechanisms for the fluorescence and the intriguingphotoluminescent properties of the BPLP polymers of the presentinvention were further explored by the inventors. As shown in FIG. 4H,monomers of citric acid, 1,8-octanediol, and L-cysteine emitted onlyvery weak autofluorescence. The POCs synthesized from citric acid and1,8-octanediol also emitted negligible photoluminescence. However, whenL-cysteine was incorporated into POC (BPLP-Cys), a strong fluorescencesignal was observed. The inventors attempted to directly synthesizepolymers from citric acid and L-cysteine or 1,8-octanediol andL-cysteine, but failed since the melting point of L-cysteine (220° C.)was much higher than the decomposition temperature of citric acid (175°C.). However, when 1,8-octanediol was reacted first with citric acid,the formed pre-POC could then dissolve L-cysteine at 160° C. to formBPLP-Cys. It is reasonable to suggest that during this synthesis theL-cysteine might be either incorporated in the pre-POC backbone orappended to the pre-POC side chains. In order to determine whichaddition was responsible for the observed fluorescence, a BPLP polymerwas synthesized in the presence of succinic acid, instead of citricacid. The resulting polymers emitted only very weak auto-fluorescence.Succinic acid is a diacid, and lacks the additional carboxylic acid andhydroxyl units found in citric acid. Thus, with succinic acid, the sideaddition of L-cysteine was not possible, supporting the hypothesis thatthe side addition of L-cysteine to citrate units was an essential stepin the formation of fluorescent polymer.

As a possible mechanism, but not a limit the inventors suggest thatL-cysteine first covalently links to the carboxylic acid on citrate toform an amide bond through its N-terminus. In a second step, the6-membered ring is formed by an esterification reaction between the freecarboxylic acid on the appended cysteine and the geminal hydroxyl unitremaining on citrate (FIG. 1A). Since all BPLPs with all 20 α-aminoacids generate significant fluorescence (Table 1), the formation of acyclic structure in this manner is consistent with the experimentaldata, regardless of the different functional units present on the aminoacid side chains.

It is well known that conjugated systems can emit fluorescence. Thefluorophore of current synthetic fluorescent polymers mostly consists ofnondegradable units containing conjugated phenyl groups or alternatingsingle and multiple bonds in the polymer chain thus makes themunsuitable for in vivo biomedical applications. It has been reported inan early work that the fluorescence of polymers may arise from theconjugated system by the cyclization of polymer side chain.⁴¹ Highlyconjugated system may emit strong fluorescence. POC and L-cysteinethemselves only emits very weak autofluorescence. The intermolecularhydrogen bonding of BPLP-cys forming conjugated 6-member ring systemsresult in the strong fluorescence emitting properties as shown in FIG.9. The 6-membered rings in the BPLPs are composed of amide and esterbonds with different pendant groups from various amino acids. Amidebonds and ester bonds are resonance stabilized so that the lone pairs onthe N and O occupy p-orbitals which conjugate with the p-orbitals on theC═O. Hyperconjugation theory²⁶ suggests that the electrons in the C—Cbond (σ-bond) at the central C3 and the C—H or C—C bond (σ-bond) at theα-C in the amino acids in the 6-membered rings can strongly associatewith p-orbitals in the neighboring C═O, N and O to extend the conjugatedsystem throughout the ring. The side chain R groups pendant to the α-Cin the amino acids likely influence the degree of hyperconjugation andpropensity for cyclization, providing slight perturbations in theassociated energy levels and resulting in the different emission maximaand quantum yields observed for the various BPLP-amino acids (Table 1).

Degradation and Mechanical Properties of BPLP Families:

The degradation rate of BPLP families was found to depend on the ratioof the monomers and the polymerization conditions (FIGS. 6A and 6B).Analysis of soluble in vitro degradation products derived from BPLP-Cysand BPLP-Ser by high performance liquid chromatography—electrosprayionization—mass spectrometry (HPLC-ESI-MS) revealed the presence of alarge amount of citrate, in addition to other soluble oligomers (FIG. 7)indicating that the primary degradation mechanism for the polymer invitro is a return to monomeric material.

FIGS. 7A and 7B shows the HPLC-ESI-MS analysis of soluble in vitrodegradation products for BPLP-Cys and BPLP-Ser degraded in 0.05 M NaOHfor 24 hours, respectively. The figures show total ion chromatograms inthe negative ionization mode from HILIC mode separation of degradationproducts. Insets show a large amount of citric acid (192 Da) is detectedas the dominant product from both polymer digests. Other signals arepreliminarily attributed to soluble oligomers. Analysis of soluble invitro degradation products (0.05 M NaOH for 24 hours) derived fromBPLP-Cys and BPLP-Ser by high performance liquidchromatography—electrospray ionization—mass spectrometry (HPLC-ESI-MS)revealed the presence of a large amount of citrate, in addition to othersoluble oligomers. The solution of degraded polymer, after filteringnonsoluble material, still exhibited significant fluorescence,indicating the presence of intact fluorophores in solution and theirincreased resistance to mild NaOH digestion. While the fluorophoreremained intact, this analysis indicated that the primary degradationmechanism for the polymer in vitro is a return to monomeric material.Taken together, these studies suggest that that Pre-POC is moresusceptible to degradation than BPLP-Ser or BPLP-Cys (at least thatportion of the polymer which contains the appended amino acid). Still,degradation in higher concentrations of NaOH for extended periods (1 MNaOH for 48 hours) did abolish fluorescence, indicating the potentialfor total degradation of the polymer under appropriate conditions.

The mechanical properties could be adjusted by varying ratios ofmonomers and by altering polymerization conditions. As shown in FIGS. 6Cand 6D, the tensile strength for CBPLP-Cys ranged from 3.25±0.13 MPa to6.5±0.8 MPa and the initial Modulus was in a range of 3.34±0.15 MPa to7.02±1.40 MPa, which were stronger than those of POC elastomers.²⁵CBPLP-Cys could be elongated up to 240±36%, which is comparable toreports of such values for arteries and veins.²⁵ The compressive modulusof BPLP-Cys (0.6) (1:1:0.6 OD:CA:Cys) scaffold was 39.60±5.90 KPaconfirming the soft nature of the scaffolds, similar to that reportedfor soft elastomers including poly(diol citrates) (POC), poly(glycerolsebacate) and xylitol-based polymers.^(25, 27-30)

Cytotoxicity Evaluation and Bioimaging Study In Vitro and In Vivo:

Cyto-compatibility of BPLPs and CBPLPs and their potential applicationsfor cellular bioimaging, drug delivery, and tissue engineering wereevaluated (FIG. 8). CBPLP-Cys films were found to support 3T3 mousefibroblast adhesion and proliferation. Viable cell numbers on CBPLPswere significantly higher than those on controls POC film andpoly(D,L-lactide-co-glycolide) (PLGA 75/25) film at day 7 (P<0.05) (FIG.8A). Importantly, cytotoxicity evaluation for degradation productssuggested that the degradation of BPLPs and CBPLPs generated similarcytotoxicity to the controls POC and PLGA75/25 (P>0.05) (FIG. 8B). Whenimplanted in vivo, the CBPLP-Ser scaffolds did not trigger noticeableedema and tissue necrosis on the tested animals. Samples that wereimplanted for 5 months produced a thin fibrous capsule, characteristicof a weak chronic inflammatory response (FIG. 9), which was expected andconsistent with the introduction of foreign materials into the body.When implanted in vivo, the polymer scaffolds did not trigger edema andtissue necrosis on the tested animals. Samples that were implanted for 5months produced a thin fibrous capsule, characteristic of a weak chronicinflammatory response, which was expected and consistent with theintroduction of foreign materials into body

Intake of BPLP-Ser nanoparticles by cells generated cells labeled withvarious fluorescence colors (FIGS. 8C to 8E). Following subcutaneousimplantation in nude mice, BPLP-Ser nanoparticles and CBPLP-Serscaffolds (FIG. 8G) were readily detected in vivo using a non-invasiveimaging system (FIGS. 8F and 8H).

The potential future applications of the unique BPLP families are worthyof further note. BPLPs can be used as fluorescence probes offeringadvantages over the traditional organic dyes and semiconductor quantumdots due to their tunable fluorescence emission, high quantum yield,degradability, photostability, and cell compatibility. The inventorshave shown in the present disclosure that BPLP nanoparticles(“biodegradable quantum dots”) can be used to label cells. Thus, it maybe possible to develop a biodegradable fluorescent drug delivery systemusing BPLPs avoiding the long-term toxicity associated with currentlabels. The low molecular weight BPLPs can be made to be water-insolubleor -soluble maximizing their potential applications in biologicallabeling and imaging. The water soluble low-molecular-weight BPLPs maypotentially be used for single molecule labeling such as protein and DNAlabeling in proteomics and genomics research, where quantum dots may notbe ideal due to their size.^(7, 8) The BPLP family may also be suitablefor use in fluorescence resonance energy transfer (FRET),⁵ two-photonexcited fluorescence microscopy,⁶ multimodal compositions (combined withmagnetic or radionuclear agents),³¹ and biosensors.³² BPLP polymersprovide real promise for non-invasive real-time monitoring of thescaffold degradation and tissue infiltration/formation in vivo, whichhas been a challenge in the evolving field of tissue engineenng.³³⁻³⁵The results obtained by the inventors have demonstrated that thefluorescent BPLP nanoparticles and CBPLP scaffolds could be imaged invivo with negligible interference from tissue autofluorescence. Inaddition the in vivo scaffold bioimaging will open new avenues for softtissue engineering studies and may provide an opportunity for doctors totrack clinical outcomes without an open surgery.

Synthesis and Characterization of BPLPs and CBPLPs:

For BPLP synthesis, equimolar amounts of citric acid and 1,8-octanediolwere combined and stirred with additional L-cysteine at molar ratios ofL-cysteine/citric acid 0.2, 0.4, 0.6, and 0.8. After melting the mixtureat 160° C. for 20 minutes, the temperature was brought down to 140° C.stirring continuously for another 75 minutes to obtain the BPLP-cysteine(BPLP-Cys) oligomers or low-molecular-weight compounds. The oligomerswere purified by precipitating the oligomer/1,4-dioxane solution inwater followed by freeze drying. Each of the 20 (L-) amino acids wasused to synthesize a family of BPLP-amino acid polymers. Water solublepolymer (BPLP-PEG-amino acid) was synthesized using poly (ethyleneglycol) (PEG), citric acid, and amino acid. Other aliphatic diols(C₃-C₁₂ diols) can also be used for BPLP synthesis similar to ourpreviously developed poly(diol citrates).²⁴ The synthesized BPLPs have ashelf-life of over a year without significant changes on theirphotoluminescent properties (emission wavelength and intensity) whenstored in amber glass bottles at −20° C. (FIG. 10). The results of thestorage stability evaluation indicates that BPLPs has shelf-life of atleast a year in terms of its photoluminescent properties when stored inamber glass bottles at −20° C.

For CBPLP film synthesis, BPLP was dissolved in 1,4-dioxane to form a 30wt. % solution and then cast into a Teflon mold followed by solventevaporation and then post-polymerization at 80° C. for 4 days. For CBPLPscaffold fabrication, a common salt-leaching method was applied.³⁶ ForBPLP nanoparticle preparation, 0.6 g of BPLP was dissolved in acetone(10 ml). The polymeric solution was added dropwise to deionized water(30 ml) under magnetic stirring (400 rpm). The setup was left overnightin a chemical hood to evaporate the acetone. TEM (JEOL-1200 EX II) anddynamic light scattering (DLS, Microtrack) were used to determine thesize, shape, and size distribution of the nanoparticles. The BPLPs werecharacterized by Fourier Transform Infrared (FT-IR), ¹H- and ¹³C-nuclearmagnetic resonance (NMR), and matrix-assisted laserdesorption/ionization mass spectroscopy (MALDI-MS; Bruker Autoflex).

Photoluminescent Properties:

Photoluminescence spectra of BPLP-Cys0.2 solutions and nanoparticles,and CBPLP-Cys0.2 films and scaffolds were acquired on a Shimadzu RF-5301PC fluorospectrophotometer. Both the excitation and the emission slitwidths were set at 1.5 nm for all samples unless otherwise stated. TheWilliams' method was used to measure the fluorescent quantum yield ofthe BPLP polymers.³⁷ The photostability of BPLP-Cys solution, BPLP-Cysfilm, BPLP-Ser solution, and Rhodamine B solution were evaluated byrecording the changes of the fluorescence intensity of the samples undercontinuous excitation in the fluorospectrophotometer. The excitationwavelength for photostability tests was determined by the maximumabsorbance spectra of each type of sample. The fluorescence changes withdegradation were determined by measuring the fluorescence intensity ofthe solutions of BPLP-Cys degraded in 0.05 M NaOH under 37° C. atvarious degradation degrees and at the same concentration.

Mechanical Tests and Degradation Studies:

The tensile mechanical tests on CBPLP films were conducted according toASTM D412a on a MTS Insight 2 mechanical tester.²⁴ The initial moduluswas measured from a slope of stress-strain curve at 10% of strain. Thecompressive tests on CBPLP scaffold (90% porosity, 100 μm pore size, 3mm height, 6 mm diameter) were conducted according to a method describedpreviously.³⁰ The in vitro degradation of BPLP and CBPLP polymers wereconducted by incubating the polymers in phosphate buffered saline(pH=7.4) at 37° C. for various times to obtain polymer mass loss.³⁶ Toanalyze the degradation products of the BPLPs, three grams of BPLPs weredegraded in 0.05 M NaOH for 24 hour and in 1 M NaOH for 48 hours.Soluble degradation products were investigated by high performanceliquid chromatography—electrospray ionization—mass spectrometry(HPLC-ESI-MS; Shimadzu LCMS-2010), using hydrophilic interactionchromatography (HILIC) on an amide-bonded stationary phase (TosohBioscience Amide-80). The filtered (0.2 μm PTFE syringe filter;Whatman), in vitro degraded sample of BPLP-Cys was analyzed to track thepresence of monomers based on retention time and mass-to-charge ratio(matched to the analysis of standards) in the negative ionization mode.

Cytotoxicity Evaluation:

Mouse 3T3 fibroblasts were used to evaluate the cytocompatibility of thepolymers. The cell viability and proliferation on CBPLP-Cys0.2 andCBPLP-Ser0.2 films (80° C., 4 d) was determined byMethylthiazoletetrazolium (MTT) assay as described previously.³⁶ Thecell morphology was observed under scanning electron microscopy (SEM,Hitachi 3500N). Cytotoxicity of the polymer degradation products wasinvestigated according to a method described elsewhere.³⁸ Briefly,BPLP-Cys, BPLP-Ser and their CBPLPs (80° C., 4 days) were hydrolyticallydegraded in 1M NaOH solution at 37° C. over a period of 24 h to 72 h.The solution was then filtered through a cellulose acetate membranesyringe filter (0.2 μm pore diameter). The pH was adjusted to 7.4 with 1M HCl. The solution was filtered again for sterilization and thendiluted by 2, 10, 50 and 100 times with culture medium. The solutionswere added to the cultured cells (n=5 wells for each polymer dilution)in 96 well plates (100 μl/well) and incubated at 37° C. and 5% CO₂ for24 h. Cell viability was then determined using MTT assay. POC (80° C., 4d) and poly(D,L-lactide-co-glycide) (PLGA75/25, Mw=113 KDa, LakeshoreBiomaterials, Birmingham, Ala.) were used as controls for the abovecytotoxicity evaluation. The statistical significance between two setsof data was calculated using a Student's t test. Data were considered tobe significant when a p value of 0.05 or less was obtained (showing a95% confidence limit).

Bioimaging Studies In Vitro and In Vivo:

For cellular fluorescence-labeling in vitro, 3T3 mouse fibroblasts wereseeded on sterile glass cover slips at a density of 5000 cells/ml for 24h prior to the cellular uptake study. The cover slips were washed withPBS and transferred to new Petri dishes, and then incubated with aBPLP-Ser0.2 nanoparticle solution in PBS (2% wt, 80 nm in diameter) for4 hours at 37° C. At the end of the study, the cells were washed (PBS×3)and then fixed with glutaraldehyde solution (2.5%). Cells were observedunder a Leica DMLP microscope (Nikon Corp. Japan). Fornanoparticle/scaffold bioimaging in vivo, BPLP-Ser0.2 nanoparticles (2%wt in PBS, 80 nm in diameter, sterilized by filtering through a syringefilter (0.22 μm)) and CBPLP-Ser0.2 scaffolds (6 mm in diameter, 90%porosity, 100 μm pore size, 1.5 mm thick, sterilized by 70% ethanol andUV light) were injected/implanted subcutaneously in nude mice (BALB/cnu/nu). The mice were then imaged using a CRi Maestro Imaging System, asdescribed previously,^(14, 39) immediately after the implantation.CBPLP-Ser scaffolds subcutaneously implanted in nude mice for 5 months(n=4) were sectioned for hematoxylin and eosin (H&E) staining topreliminarily evaluate the long-term in vivo host responses to thepolymers.

To non-invasively monitor the scaffold degradation in vivo, it isimperative to understand the relation of scaffold fluorescenceproperties and the scaffold degradation over time in vitro. Thefluorescence of polymers is proportional to the concentration of thepolymers. The scaffold degradation may cause the changes of fluorescenceintensity, wavelength, and size of fluorescent scaffold. Therefore, thepresent inventors established a novel non-invasive fluorescence basedbioimaging method to study the scaffold degradation and tissue/scaffoldinteraction in vitro and in vivo using a cutting-edge CRI MaestroFluorescence Imaging system.

The present inventors have also devised methods for synthesizingcrosslinked urethane-doped BPLP (CUBPLP), water-soluble BPLP (WBPLP),and photo-crosslinkable BPLP (PCBPLP). The methods of synthesis andcharacteristics of the BPLP variants is described below:

Crosslinked Urethane-Doped BPLP (CUBPLP):

CUBPLP synthesis was carried out in three steps. Step 1, citric acid and1,8-octanediol, with a monomer ratio of 1:1.1, were bulk polymerized ina round bottom reaction flask. After the monomers had melted at 160° C.,the temperature was lowered to 140° C., and the reaction mixture wasstirred for another 20 minutes to create the oligomer ofpoly(octamethylene citrate). Then additional L-cysteine at molar ratiosof L-cysteine/citric acid 0.2, 0.4, 0.6, and 0.8 was added into themixture and stirred for another 60 minutes. The pre-BPLP-Cys waspurified by drop wise precipitation in deionized water. Undissolvedpre-BPLP-Cys was collected and lyophilized to obtain the purified BPLPprepolymer. The average molecular weight of pre-BPLP-Cys wascharacterized as 1300 Da by Matrix assisted laser desorption/ionizationmass spectroscopy (MALDI-MS) using an Autoflex MALDI-TOF MassSpectrometer (Bruker Daltonics, Manning Park, Mass.). Chain extension ofthe BPLP pre-polymer to obtain pre-CUBPLP was done in step 2. Purifiedpre-BPLP-Cys was dissolved in 1,4-dioxane to form a 3% (wt/wt) solution.The polymer solution was reacted with 1,6-hexamethyl diisocyanate (HDI)in a clean reaction flask under constant stirring, with stannous octoateas catalyst (0.1% wt). Different pre-CUBPLP polymers were synthesizedwith different feeding ratio of pre-BPLP:HDI (1:0.5, 1:1.0, and 1:1.5,molar ratio) and different amino acids. The system was maintained at 55°C. throughout the course of the reaction. Small amounts of the reactionmixture were removed at 6 hour intervals and subjected to Fouriertransform infrared (FT-IR) analysis. The reaction was terminated whenthe isocyanate peak at 2267 cm⁻¹ disappeared. In step 3, the pre-CUBPLP(UBPLP) solution was cast into a laminar airflow until all the solventshad evaporated. The resulting UBPLP film was moved into an ovenmaintained at 80° C. for pre-determined time periods to obtaincrosslinked urethane-doped polyester (CUBPLP). The excitation and theemission spectra of the synthesized UBPLP-Cys are shown in FIG. 11. Thequantum yield of UBPLP-Cys0.2 was 36%. The tensile strength ofCBPLP-Cys0.2 was as high as 40 MPa with 275% elongation (FIGS. 12 and13).

Water-Soluble BPLP (WBPLP):

For Water-soluble BPLP (WBPLP) synthesis, citric acid and poly(ethyleneglycol) at molar ratio of 1:1.1 were combined and stirred withadditional L-cysteine at molar ratios of L-cysteine/citric acid 0.2,0.4, 0.6, and 0.8. After melting the mixture at 160° C. for 30 minutes,the temperature was brought down to 140° C. stirring continuously foranother 8 h to obtain the WBPLP-cysteine (WBPLP-Cys) linear prepolymer.The prepolymers were purified by dialysis against deionized water usingdialysis bag with molecular weight cut off of 500 Da. The DI water waschanged every 6 hours. After one week, the polymer solution was freezedried. Each of the 20 (L-) amino acid was used to synthesize a family ofWBPLP-amino acid polymers. The quantum yields of WBPLP-Cys0.2 andWBPLP-Ser0.2 were 40% and 22% respectively. The excitation and emissionspectra of WBPLP-PEG200-Cys0.2 and WBPLP-PEG200-Ser0.2 are shown inFIGS. 14 and 15 respectively,

Photo-Crosslinkable BPLP (PCBPLP):

The Photo-crosslinkable BPLP was synthesized as following steps. First,citric acid, maleic acid, and 1,8-octanediol were added into a roundbottom reaction flask with molar ratio of 0.2:0.8:1.1. The mixture wasmelted under 160° C. Additional amount of L-Cysteine with molar ratiosof L-cysteine/acid 0.2, 0.4, 0.6, and 0.8 was added into reaction systemand stirred continuously for another 6 hours to obtain thePCBPLP-cysteine. The PCBPLP-cysteine was purified by drop wiseprecipitation in deionized water. After purification, the prepolymer wasthen lyophilized for further characterization. FIG. 16 shows theexcitation and emission spectra of PCBPLP-MA0.8-Cys0.2. The family ofPCBPLP was obtained by using 20 (L-) amino acid for synthesis.

The present inventors also fabricated tubes and soft and elastic CBPLPSDBV scaffolds via a scaffold-sheet tissue engineering strategy. FIGS.17A and 17B shows the tubes fabricated from the BPLP of the presentinvention under natural light and UV light, respectively. In order toassess whether the CBPLP scaffold-sheet SDBV graft design can modulatecell-cell communication, cell proliferation, cell differentiation, cellmigration and matrix production for SDBV regeneration was studied in anin vitro in a perfusion bioreactor via histological analysis andbioimaging tool.

The feasibility of growing small diameter blood vessels (SDBV) in vitroin a bioreactor using cell-seeded CBPLP grafts was studied. The anatomyof artery shows that the elastic lamina separates the endothelium andthe medium layer (smooth muscle cell (SMC) layer) surrounded byadventitial layer (fibroblasts). In vivo, each cell types of thevascular wall is “dependent” on its neighboring cells, and all actsynergistically toward the development, maintenance, remodeling, andregulation of the tissue under physiological and pathologicalconditions. Interaction between endothelial cells (ECs) and SMCs wasbelieved to be important in determining vessel diameter, thickness, SMCproliferation and phenotype via either physical contact or theirsecreted soluble biological mediators such as transforming growthfactor-1 (TGF-1).⁴² Many studies have focused on the SMC-ECcommunications. However, the fibroblast cell seeding was not includeddue to the incapability of seeding the third cell types in thosescaffold designs.⁴³⁻⁴⁸ There was no any scaffold design that couldaddress all the concerns of the compartmentalization of the three typesof cells (fibroblasts, SMCs and ECs) without hampering the cell-cellcommunication between SMCs and ECs, the uneven cell distribution, thecompliance mismatch, and the off-the-shelf availability for in vivotissue engineering SDBVs. The inventors assessed whether the CBPLPscaffold-sheet graft can serve as a model to study the SMC-ECcommunications in existence of fibroblasts, cell migration,differentiation and matrix production in vitro via a perfusionbioreactor. The fluorescent CBPLP SDBV grafts were also characterized bythe Maestro Imaging System to monitor the scaffold degradation andtissue regeneration.

Scaffold-Sheet Fabrication:

As previously shown by the inventors the poly(ethylene glycol) dimethylether (PEGDM) can be used as a porogen to create tortuous nano- orsub-micro channels in the POC films,⁴⁹ a permeable CBPLP-ser tube(50-100 μm of thickness, 3 mm in diameter) was fabricated by dip-coatinga glass rod (3 mm in diameter) into the BPLP/PEGDM (Mw=400, 20 wt % ofBPLP-ser) solution followed by solvent evaporation, postpolymerization,PEGDM leaching, and then freeze-drying.

The permeability of the tubes was tested according to the methoddescribed previously.⁵⁰ Thin CBPLP porous scaffold sheets (100 μm thick,100 μm of pore size) were fabricated via a thermally inductive phaseseparation method by freeze-drying BPLP-ser solution in a teflon moldfollowed by post-polymerization in an oven (80° C., 4 days). Themacropore structure and nanoporous features of CBPLP-ser scaffold andlumen tubes were observed by high resolution SEM.

Construction of SDBV Graft Using Cell-Seeded Fluorescent CBPLP-SerScaffold Sheets:

6 CBPLP-ser scaffold sheets and one permeable CBPLP-ser tube were usedfor cell seeding and fabrication of the tubular SDBV graft (FIG. 18).Scaffold sheet 1-3 was be seeded with human aortic fibroblasts (HAFBs)by pipetting 5×106 cells/ml of HAFBs evenly into the scaffold; Scaffoldsheet 4-6 were seeded with human aortic smooth muscle cells (HASMCs) bypipetting 5×106 cells/ml of HASMCs evenly into the scaffold. After 2days of in vitro culture, a CBPLP SDBV graft was constructed by rollingHASMCs seeded CBPLP-ser sheets and HAFBs seeded CBPLP-ser sheets on apermeable CBPLP-ser tube sequentially on the Teflon rod (3 mm indiameter). After the rod removal, then human aortic endothelial cells(HAECs) with a density of 1×10⁶ cells/ml were seeded on the lumen of thetubular graft according a method described previously.²⁹ The resultingcell-seeded CBPLP graft will be further cultured in vitro for 3 days ina coculture medium⁴⁵ for further bonding between the layers of the graftbefore it is assembled in a perfusion bioreactor.

Tissue Culture in a Perfusion System:

A custom-made perfusion bioreactor chamber was assembled into a similarclose-loop flow system.⁵¹ A LED micrometer (Keyence LS7000) was used tonon-invasively measure radial distension imparted to the engineeredvessels. The pressure and distension data over time were recorded withLabVIEW software (National Instruments, Austin Tex.). Percent distentionwas calculated from the maximum and minimum diameters[((Dmax−Dmin)/Dmin)×100]. Blood vessel graft compliance was be obtainedby dividing the % distention by the measured pressure.

The whole system was set up in a standard cell culture incubator. TwoCBPLP grafts were assembled in one bioreactor chamber. Three bioreactorchambers were juxtaposed into the close-loop system for the followingstudies. CBPLP grafts were constructed as described above. Two graftsper chamber were mounted on the hollow posts. For cell-cellcommunication study, 6 fully seeded grafts with permeable lumen tube(fibroblasts, SMCs and ECs), 6 partially seeded CBPLP graft withpermeable lumen tube (SMCs and ECs), and 6 fully seeded CPEU grafts withnon-permeable lumen tube (without using PDMEG as nanoporogen) were used.The cell culture medium flow was kept at 40 ml/min and at a pulsefrequency of 1.5 Hz by the peristaltic pump^(.[45, 52]) The above threetypes of grafts were simultaneously cultured for 3 days, 15 days, 28days, respectively (2 grafts per time points for each type of graft).The compliance changes over time of the CBPLP grafts were monitored atthe above 3 time points to determine whether the grafts can maintain thecompliance during the matrix productions before the grafts are harvestedfor the following characterizations. The tissue constructs wereharvested from the bioreactor and imaged and characterized by the CRIMaestro Imaging system. The grafts were sectioned for biologicalanalysis to study the matrix. production (total collagen assay,⁵³elastin assay, sulfated glycosaminoglycan assay⁵⁴); matrix distributionand cell differentiation status (H&E staining for overall cell andmatrix distribution, Masson's trichrome staining for collagen, Verhoff'sstaining for elastin, calponin, actin, myosin heavy chain staining forhuman aortic smooth muscle cells (HASMC) differentiation, and vWF andVE-cadherin for human aortic endothelial cell (HAEC)differentiation).^([29,30, 55, 56]) SEM observation was performed on thelumen to assess the EC alignment. EC retention under the shear stresswas also assessed. The non-cell-seeded (dry) and cell-seeded (wet) CBPLPgrafts were subjected to the following characterization: Burstpressure,³⁰ and Suture retention.^([57, 58]) Quantitative data wasanalyzed using GraphPad Prism (4.0) with one-way analysis of variance ortwo-tail Student's t-test. Data was taken to be significant when a Pvalue of 0.05 or less is obtained.

The present inventors have developed a convenient fluorometric methodfor accurate polyquaternium-1 determination using strongfluorescence-shining polymers. Polyquaternium-1 is an antibacterialpreservative in pharmaceutical formulations. Therefore, the accuratedetermination of polyquaternium-1 is a key for quality control ofproducts and is also vital to be able to pass the Food and DrugAdministration (FDA) regulatory scrutiny.

Polyquaternium-1,ω-{4-[tris(2-hydroxyethyl)ammonio]-but-2-enylpoly(dimethylammoniobut-2enyl)}tris(2-hydroxyethyl)ammoniumpolychloride as shown in FIG. 19, is a cationic polymer with molecularweight ranging from 5,000 to 10,000. Polyquaternium-1 does not have achromophore that absorbs above 200 nm. Therefore, polyquaternium-1, byitself, cannot be detected by spectrophotometry. The theory behind thespectrophotometric determination was that an anionic organic dye, trypanblue form a water-soluble ion pair with cationic polyquaternium-1. Theformation of the water-soluble complex between polyquaternium-1 andtrypan blue increases the electron delocalisation in trypan blue andproduce a bathochromic shift of trypan blue absorbance from 658 to 700nm. Thus, the absorbance of the complex can be measured at its maximumdifference (ΔA) from a blank at 680 nm.

The introduction of trypan blue brings a chromophore kelated withpolyquaternium-1 thus provide a solution to detect the quaterniumspectrophotometrically. However, there are some drawbacks which bringabout the concerns on the sensitivity of the determination. Althoughthis method measures the absorbance at the wavelength (680 nm) ofmaximum difference between the water soluble complex and trypan blue,the interference of background signal from trypan blue is inevitablesince trypan blue also absorbs at 680 nm significantly. The absorbancefor analytes may be very low, especially when the concentration ofsamples is low (10 ppm for polyquaternium-1) plus the background signalsneed to be subtracted. Thus the sensitivity of the measurement will beaffected. In addition, the absorbance of samples is also affected by thepH value and ionic strength of sample solution which brings morevariables to the measurements. These are intrinsic problems ofabsorbance measurement. The complex can be only stable for a certainperiod of time. Thus, the stability of the ion pairs is also a concernwhich may also affect the sensitivity of the determination. The lowsensitivity may affect the repeatability of the absorbance measurements

In conclusion, in order to detect polyquaternium-1, a chromophore (dye)can be kelated with polyquaternium-1 to confer the detectability. Butthe relatively strong background signal, effects of solution pH andionic strength, stability of complex, and weak absorbance in absorbanceanalysis may affect the sensitivity of the determination.

The present invention describes a fluorometric method to accuratelydetermine the concentration of polyquaternium-1. Given the fact thatpolyquaternium-1 is a positively charged polymer with molecular weightfrom 5000 to 10000, a negatively charged polymer should be able tokelate with polyquaternium-1 to form a complex. The stability of theformed ion pair complex may affect the sensitivity of the fluorometricanalysis. The precursors of BPLPs are highly negatively chargedpolymers. The introduction of various amino acids may not only conferfluorescence to BPLP but also modulate the charge state of the polymers.Unlike trypan blue which is a small molecule and consists of mainlystiff benzene ring, the polymer backbone of BPLPs is a flexiblepolyester chain. The chain length of BPLP is also much longer than thatof the trypan blue. It is expected that the long (around 2000 Dalton)and flexible negatively charged BPLP chains should entangle with longand positively charged polyquaternium-1 chain to form a stable ion paircomplex. The entanglement should enhance the stability of the ion paircomplex thus may improve the sensitivity of determination.

Fluorescence signal is proportional to the concentration of analytes. Astrong fluorescence dye is preferred to detect the low concentration ofanalytes. Compared to the other commercially available organicfluorescence dyes, BPLPs are much stronger fluorescent polymers whichamplify the signals thus improve the sensitivity of determination.

BPLPs consist of water-soluble and solvent soluble polymers. Both typesof BPLP polymers were tested for polyquaternium-1 determination. Whenwater-soluble BPLPs are hybrid with polyquaternium-1, the formed complexmay be both water soluble complex and water-insoluble. a shift offluorescence emission wavelength. If the complex is water-insoluble, thecomplex will be isolated from the aqueous solution first and thenre-suspended in water or a organic solvent for fluorescencemeasurements. A standard curve will be established using a series ofknown concentration of polyquaternium-1. If the complex is stillwater-soluble, the protocol of trypan blue method was followed.

If water-insoluble BPLPs are used, BPLP organic solutions will be mixedwith polyquaternium-1 aqueous solution. The kelating process may resultin the transfer of polyquaternium-1 from water phase into organic phase.The organic phase was collected to do the fluorescence measurements. Ifthe complex remains in the water phase, the water phase was collectedfor fluorescence measurements. To detect which phase the complex stays,FTIR analysis or HPLC was performed.

For water soluble complex, alternatively, HPLC was used to obtain pureBPLPpolyquaternium-1 complex which was be subjected to the fluorescencemeasurements. The isolation was obtainable since the size of the complexshould be different from both BPLP and polyquaternium-1. Dialysis couldbe used to purify the BPLPpolyquaternium-1.

The present invention describes the discovery of a family of aliphaticbiodegradable photoluminescent polymers (BPLPs and CBPLPs) that emittunable, strong, and stable fluorescence. The synthesis of BPLPs andCBPLPs was straightforward and cost-effective. BPLP families possessexcellent processability for micro/nano fabrication and desiredmechanical properties, potentially serving as implant materials andbioimaging probes in vitro and in vivo. The data shows that the CBPLPssupport cell attachment in vitro and only exert weak chronicinflammation in vivo. The development of BPLPs and CBPLPs represent anew direction in developing biodegradable materials and may have wideimpact on basic sciences and a broad range of applications such astissue engineering, drug delivery, and bioimaging.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method, kit, reagent, orcomposition of the invention, and vice versa. Furthermore, compositionsof the invention can be used to achieve methods of the invention.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, numerous equivalents to the specificprocedures described herein. Such equivalents are considered to bewithin the scope of this invention and are covered by the claims.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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The invention claimed is:
 1. A polymer composition comprising: anoligomer synthesized from monomers comprising (i) a multifunctionalmonomer comprising citric acid or triethyl citrate, (ii) a diol, and(iii) an amino acid, wherein the amino acid is linked as a side group tothe oligomer backbone.
 2. The composition of claim 1, wherein the diolis selected from 1,8-octanediol, ethylene glycol, propylene glycol,poly(ethylene glycol), polypropylene glycol), 1,3-propanediol,ethanediol, and cis-1,2-cyclohexanediol.
 3. The composition of claim 1,wherein the amino acid comprises alanine, arginine, asparagine, asparticacid, cysteine, glycine, glutamine, glutamic acid, histidine,isoleucine, leucine, lysine, methionine, proline, phenylalanine, serine,threonine, tyrosine, tryptophan, valine, or a combination thereof. 4.The composition of claim 1, wherein the amino acid comprises an L-aminoacid, D-amino acid, D,L-amino acid, or a derivative thereof.
 5. Thecomposition of claim 1, wherein an acid anhydride or a multifunctionalacid chloride is used in addition to the citric acid or triethylcitrate.
 6. The composition of claim 1, wherein maleic acid, maleicanhydride, fumaric acid, fumaryl chloride, acryloylchloride, itaconicacid, or allylmalonic acid is used in addition to the citric acid ortriethyl citrate.
 7. The composition of claim 1, wherein the oligomer iscrosslinked.
 8. A composition comprising: an oligomer synthesized frommonomers comprising (i) a multifunctional monomer comprising citric acidor triethyl citrate, (ii) a diol, (iii) an amino acid, and (iv) adi-isocyanate wherein the amino acid is linked as a side group to theoligomer backbone.
 9. The composition of claim 8, wherein themultifunctional monomer comprises citric acid.
 10. The composition ofclaim 8, wherein the diol is selected from 1,8-octanediol, ethyleneglycol, propylene glycol, poly(ethylene glycol), poly(propylene glycol),1,3-propanediol, ethanediol, and cis-1,2-cyclohexanediol.
 11. Thecomposition of claim 8, wherein the amino acid comprises alanine,arginine, asparagine, aspartic acid, cysteine, glycine, glutamine,glutamic acid, histidine, isoleucine, leucine, lysine, methionine,proline, phenylalanine, serine, threonine, tyrosine, tryptophan, valine,or a combination thereof.
 12. The composition of claim 8, wherein theamino acid comprises an L-amino acid, D-amino acid, D,L-amino acid, or aderivative thereof.
 13. The composition of claim 8, wherein themultifunctional monomer comprises citric acid, the diol comprises1,8-octanediol, the amino acid comprises cysteine or serine, and thedi-isocyanate comprises hexamethylene-1,6-di-isocyanate or1,4-butane-di-isocyanate.
 14. A method of making a crosslinkedurethane-doped biodegradable photoluminescent polyester (CUBPLP)comprising: mixing (i) a multifunctional monomer comprising citric acidor triethyl citrate and (ii) a diol to form a mixture; raising thetemperature of the mixture to melt the mixture; lowering the temperatureof the mixture with stirring to form an oligomer; adding an amino acidto the oligomer with stirring to form a pre-BPLP-amino acid; purifyingthe pre-BPLP-amino acid; dissolving the purified pre-BPLP-amino acid toform a solution; adding a di-isocyanate to the pre-BPLP-amino acidsolution to form a pre-CUBPLP (UBPLP); casting a film of the pre-CUBPLP(UBPLP); and drying the pre-CUBPLP (UBPLP) film to obtain the CUBPLP.15. The method of claim 14, wherein the diol is selected from1,8-octanediol, ethylene glycol, propylene glycol, poly(ethyleneglycol), poly(propylene glycol), 1,3-propanediol, ethanediol, andcis-1,2-cyclohexanediol.
 16. The method of claim 14, wherein themultifunctional monomer comprises citric acid, the diol comprises1,8-octanediol, the amino acid comprises cysteine or serine, and thedi-isocyanate comprises hexamethylene-1,6-di-isocyanate or1,4-butane-di-isocyanate.
 17. The method of claim 14, wherein purifyingthe pre-BPLP-amino acid comprises: adding the pre-BPLP-amino acid todeionized water; collecting an undissolved pre-BPLP-amino acid portionfrom the deionized water; and lyophilizing the collected pre-BPLP-aminoacid to obtain purified pre-BPLP-amino acid.
 18. The method of claim 14,wherein dissolving the purified pre-BPLP-amino acid to form a solutioncomprises dissolving the purified pre-BPLP-amino acid in 1,4-dioxane.19. The method of claim 14, wherein the film of the pre-CUBPLP (UBPLP)is cast in a laminar airflow.
 20. The method of claim 14, whereinforming the pre-BPLP-amino acid solution further comprises adding one ormore catalysts to the solution, the one or more catalysts being selectedfrom tin octanoate, dibutyl tin dilaurate, and1,4-diazabicyclo[2.2.2]octane.